Multiscale Modeling of Protein Kinase Structure, Catalysis and Allostery PROJECT SUMMARY/ABSTRACT The long-term goal of this research is to advance our understanding of the catalytic and regulatory mechanisms of complex enzymatic systems and the roles of protein dynamics in enzyme function. Protein kinase (PK), the focus of this study, is an attractive system for this purpose, because it involves both large-scale conformational change and enzymatic catalysis. Moreover, because of its pathological significance, understanding PK?s molecular mechanism is of fundamental importance in kinase research and also may provide new insights into the development of improved therapies against kinases. Our central hypothesis, based on our recent studies and enzyme kinetic data, is that the catalytic activity of PK is closely associated with its regulatory function. Therefore, any change in regulatory activity affects the catalytic activity of the kinase, which occurs through allosteric modulation of underlying protein dynamics, and together control overall activity of PK. This contrasts with the conventional view that the inactive-to-active conformational change is the main mechanism for regulating kinase activity. Our objective in this grant is to examine these two contrasting views on the regulation of kinase activity by the parallel study of two important kinases, insulin receptor kinase (IRK) and adenylate kinase (AdK), which play critical roles in cell homeostasis, and elucidate their complete molecular mechanisms. These objectives will be accomplished through quantitative modeling of their conformational change, ligand binding and catalysis in key functional states, including the fully active and inactive states. The proposed research involves development of new multiscale simulation methods combining quantum, molecular and statistical mechanical methods and their extension to permit rapid and accurate determination of kinase mechanisms. Our specific AIMs are: (1) the development of effective multiscale simulation methods integrating the ab initio/density functional theory (AI/DFT) and semiempirical (SE) QM/MM methods with the string simulation methods in CHARMM, their acceleration through advanced parallelization and accelerator algorithms and reaction-specific parameterizations, and development of efficient alchemical free energy simulation methods overcoming the limitations of existing methods; 2) elucidation of the mechanisms of IRK catalysis and conformational change and the connection between its intrinsic protein motions and catalysis of IRK; and 3) determination of the catalytic mechanism of AdK and the role of active site residues in controlling the active site and global protein dynamics and the overall activity of AdK. The completion of the proposed study will deepen the mechanistic understanding of these kinases and the role of protein dynamics in their catalysis. Experimental verification of computational results will also be made by characterization of their kinetic and structural parameters via collaboration with an experimental group. Finally, the theoretical methods developed are general and can be applied readily to numerous enzymatic systems involving conformational changes and catalysis, such as ATP/GTPases and various motor proteins.